Aging of Plant

PP70CH15_Nam ARjats.
cls February 16, 2019 14:4
Annual Review of Plant Biology

Leaf Senescence: Systems and
Dynamics Aspects
Hye Ryun Woo,1,∗ Hyo Jung Kim,2,∗ Pyung Ok Lim,1,†
Access provided by Washington University - St. Louis on 02/27/19. For personal use only.
and Hong Gil Nam1,2,†

Annu. Rev. Plant Biol. 2019.70. Downloaded from www.annualreviews.org
1
Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology
(DGIST), Daegu 42988, Republic of Korea; email: polim@dgist.ac.kr, nam@dgist.ac.kr
2
Center for Plant Aging Research, Institute for Basic Science (IBS), Daegu 42988,
Republic of Korea
Annu. Rev. Plant Biol. 2019. 70:15.1–15.30 Keywords

The Annual Review of Plant Biology is online at
chloroplasts, environmental stresses, leaf senescence, nutrient
plant.annualreviews.org
remobilization, plant hormones, plant productivity, spatiotemporal
https://doi.org/10.1146/annurev-arplant-050718-
regulatory networks
095859
Copyright © 2019 by Annual Reviews. Abstract

All rights reserved
Leaf senescence is an important developmental process involving orderly
∗
These authors contributed equally to this article
†
disassembly of macromolecules for relocating nutrients from leaves to other
Corresponding authors
organs and is critical for plants’ fitness. Leaf senescence is the response of
an intricate integration of various environmental signals and leaf age in-
formation and involves a complex and highly regulated process with the
coordinated actions of multiple pathways. Impressive progress has been
made in understanding how senescence signals are perceived and processed,
how the orderly degeneration process is regulated, how the senescence
program interacts with environmental signals, and how senescence regula-
tory genes contribute to plant productivity and fitness. Employment of sys-
tems approaches using omics-based technologies and characterization of key
regulators have been fruitful in providing newly emerging regulatory mech-
anisms. This review mainly discusses recent advances in systems understand-
ing of leaf senescence from a molecular network-dynamics perspective. Ge-
netic strategies for improving the productivity and quality of crops are also
described.
15.1
Review in Advance first posted on
February 27, 2019. (Changes may
still occur before final publication.)
PP70CH15_Nam ARjats.cls February 16, 2019 14:4
Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
MULTIFACETED CHANGES IN CHLOROPLASTS AND MITOCHONDRIA
DURING LEAF SENESCENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Regulation of Chlorophyll Breakdown and Chloroplast
Degeneration During Leaf Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
The Morphological and Metabolic Changes of Mitochondria in Leaf Senescence . . . 6
THE FUNCTIONAL AND REGULATORY TRANSITIONS
IN LEAF SENESCENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
The Temporal Dynamics and Functions of Gene Regulatory Networks
Involving NAC Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
The Dynamics and Functions of WRKY Gene Regulatory Networks Underlying

Leaf Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Omics Approaches for Temporal Profiling of Molecular Processest

in Leaf Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
MULTILAYERED REGULATION OF STRESS-INDUCED LEAF
SENESCENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
The Chromatin-Mediated Regulation of Stress-Induced Leaf Senescence . . . . . . . . . . 11
The Transcriptional Regulation of Stress-Induced Leaf Senescence . . . . . . . . . . . . . . . . 12
Posttranscriptional Regulation of Stress-Induced Leaf Senescence . . . . . . . . . . . . . . . . . 13
Posttranslational Regulation of Stress-Induced Leaf Senescence . . . . . . . . . . . . . . . . . . . 13
CROSSTALK BETWEEN HORMONE SIGNALING
AND LEAF SENESCENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
OTHER EMERGING REGULATORY MECHANISMS OF LEAF
SENESCENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Light Signaling and Leaf Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Circadian Rhythm, Aging Clock, and Leaf Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Age Gating: Function of a Senescence Regulatory Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
AGRONOMIC IMPLICATION OF LEAF SENESCENCE RESEARCH . . . . . . . . . . 20
Development of Strategies for Efficient Nitrogen Utilization . . . . . . . . . . . . . . . . . . . . . . 20
Manipulation of Key Regulatory Genes Associated with the External
and Internal Factors that Affect Leaf Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
INTRODUCTION
Aging is by definition an addition of age, which leads to age-dependent deterioration or senes-
cence of cells, tissues, organs, and organisms at later stages of life history in both animals and plants.
Meristematic tissue:
tissue that has the However, the term senescence is used mostly in plants for deteriorative and degenerative physio-
ability to enlarge, logical changes and in animals for mitotic or replicative senescence of cells. Plants show two types
stretch, and of senescence: mitotic or replicative senescence and postmitotic senescence (25). Mitotic senes-
differentiate into other cence is exhibited in meristematic tissue following the arrest of cell division or replication activity.
types of cells as it
Postmitotic senescence occurs in plant organs such as leaves and floral petals after their cellular
matures
differentiation and maturation has completed and accompanies an active and programmed degen-
erative process (70). Senescence in plants occurs at various levels but most conspicuously at the
15.2 Woo et al.

organ and organismal levels, as illustrated by the splendid autumn scenery of the color changes of
leaf organs.
The leaf is the primary photosynthetic organ for energy harvesting and nutrient production
Annual plant: a plant
at the growth and maturation stages. When a leaf enters the senescence stage, its cells undergo that performs its entire
the sequential disorganization of cellular organelles and orderly changes in metabolism and gene life cycle from
expression (Figure 1). Major transitions during leaf senescence are the breakdown of chloroplasts germination to the
and the replacement of carbon assimilation by the catabolism of chlorophyll and macromolecules. production of seeds
within a single
Increased catabolic activity is responsible for converting cellular materials accumulated during
growing season
the leaf’s growth phase into exportable nutrients. In annual plants, disassembled nutrients are
supplied to developing seeds as a parental investment. In perennial plants, such as trees, nutrients Perennial plant:
a plant that persists for
are relocated to the stems or roots, which are used as storage resources for the development of new
more than two years
leaves or flowers in the next season. Thus, leaf senescence is a critical process for ensuring optimal
production of offspring and better survival. The degeneration and remobilization processes, which Multi-omics:
biological analysis
occur concomitantly, are therefore likely to be orderly and highly coordinated (10, 68).
approach in which the
Leaf senescence spans the latter half of the leaf’s life and thus constitutes a significant portion data sets are multiple
of its life history. Leaf senescence is basically controlled by developmental age. However, leaf “omes”, such as
senescence also proceeds by integrating various internal and external environmental signals into genomes, epigenomes,
age information (10, 68) (Figure 1). These include nutritional and hormonal signals, water status, transcriptomes,
proteomes, and
light regimes, and temperature change. Thus, the interconnection of highly intricate regulatory
microbiomes
networks and multiple, crosstalking pathways should be operating during leaf senescence, and
these networks should be dynamic to regulate and reflect the senescence state affected by various Shelf life: period that
a commodity remains
senescence signals (68).
effective and free from
In this review, we first briefly describe the molecular changes of leaf senescence and then dis- deterioration, without
cuss some recent results that explore new regulatory mechanisms identified by use of multi-omics becoming unfit for use,
technologies with computational biology and imaging tools and extensive molecular genetic anal- consumption, or sale
yses. We address the rapid identification of novel regulatory molecules and their regulatory net-
works, elucidation of the spatiotemporal dynamics of age-associated networks, and the exploration
of molecular mechanisms that integrate various environmental signals with senescence pathways
(Supplemental Table 1). Furthermore, the current progress that translates basic research findings
into practical applications for enhancing the productivity and quality of crops and/or prolonging
the shelf life of horticulture products is overviewed.
MULTIFACETED CHANGES IN CHLOROPLASTS AND

MITOCHONDRIA DURING LEAF SENESCENCE
Leaf cells are subject to massive structural and biochemical changes in an orderly manner during
senescence (68). One of the most distinctive characteristics of biochemical changes during leaf
senescence is the dramatic metabolic transition from anabolism to catabolism, including the in-
creased hydrolysis of macromolecules (Figure 1). The following section summarizes the state of
knowledge about the cellular structural and biochemical changes during leaf senescence and de-
scribes the underlying molecular regulatory mechanisms, particularly with regard to chloroplasts
and mitochondria.
Regulation of Chlorophyll Breakdown and Chloroplast

Degeneration During Leaf Senescence
The fist visible phenotypic change in leaf senescence is the color change of the leaf due to the
preferential breakdown of chlorophyll concomitant with chloroplast disassembly. Chlorophyll
www.annualreviews.org • Leaf Senescence 15.3

a Organ Sexual
development reproduction
Cell wall biogenesis Cell wall disassembly

Biological process
Carbohydrate/
Macromolecule biosynthesis lipid catabolism
Photosynthesis
Anthocyanin biosynthesis/
Cell cycle amino acid transport
Chromatin assembly/DNA replication

GA/cytokinin response
JA/ABA response
SA response
emergence
Days after
4 6 8 10 12 14 16 18 20 22 24 26 28 30
b ANAC083 ANAC017 ANAC082 ANAC090 ANAC055 ANAC090 ANAC076 ANAC079
007 016 019 032 001 002 004 016 019 001 016 019 021 029 019 041 042 046 047
041 042 053 061 087 032 041 042 046 047 048 041 042 046 047 053 055 061 072 074
053 055 056 059 061 056 061 079 081 084 084 087 100 104 12910*
090 100 102 104
081 087 096 100 102 12910* 087 100 104 12910*
14 days 18 days 22 days 26 days
after emergence after emergence after emergence after emergence
TRANSITION OF REGULATORY NET WORK
c Developmental
age
Endogenous Exogenous
factors factors
(Caption appears on following page)
15.4 Woo et al.

Figure 1 (Figure appears on preceding page)

Overview of a leaf life history. (a) Leaves undergo developmental transitions throughout their life history. (b) Many molecular
components during leaf senescence show complex interactions, and their functional and regulatory interaction networks change
continuously with each progressive stage of senescence. One representative example is a temporal network dynamic involving NAC
family transcription factors during leaf senescence. In each subnetwork, circles represent target NACs regulated by the corresponding
hub NACs, and blue and orange lines indicate positive and negative regulations of target NACs by the hub NACs. Accordingly, leaf
cells undergo dramatic shifts in physiology from biogenesis to the sequential degeneration of macromolecular and cellular structures,
including the breakdown of the photosynthetic machinery in a coordinated manner. 12910* refers to At3g12910. Panel b adapted from
Reference 49. (c) Leaf senescence, the last stage of leaf development, is tightly regulated by developmental age, but it is also influenced
by various endogenous and exogenous factors that are integrated into age information. Abbreviations: ABA, abscisic acid; ANAC,
ABSCISIC ACID–RESPONSIVE NAM/ATAF/CUC; GA, gibberellin; JA, jasmonic acid; NAC, NAM/ATAF/CUC; SA, salicylic acid.
breakdown is governed by the pheophorbide a oxygenase (PAO)/phyllobilin pathway, which

comprises of a set of chlorophyll catabolic genes (CCGs) (40). This process is initiated by
conversion from chlorophyll b to chlorophyll a by chlorophyll b reductase, which is encoded
by NONYELLOW COLORING1 (NYC1) and NONYELLOW COLORING1–LIKE (NOL), and

7-hydroxymethyl chlorophyll a reductase (HCAR). Magnesium is removed from chlorophyll
a to be converted to pheophytin a by magnesium-dechetalase encoded by NONYELLOW-
INGs/STAYGREENs (NYEs/SGRs). Pheophytin a is then hydrolyzed by PHEOPHYTINASE
(PPH) to produce pheophorbide a and phytol. Notably, the green color of chlorophyll catabolites
is completely lost when the porphyrin ring of pheophorbide a is cleaved by PAO, resulting in
oxidized red chlorophyll catabolite, which is subsequently catalyzed by red chlorophyll catabolite
reductase to generate primary fluorescent chlorophyll catabolite (pFCC). Finally, pFCC is mod-
ified, transported into the vacuole, and isomerized to nonfluorescent products by acidic pH (40).
Recent studies have taken advantage of the well-characterized CCGs to uncover the regulatory
mechanisms that control chlorophyll breakdown (57).
Particularly illuminating was the transcriptional regulation in chlorophyll breakdown that
emerged from studies of Arabidopsis in recent years (Figure 2). Two Arabidopsis basic leucine zip-
per (bZIP) transcription factors (TFs), ABSCISIC ACID (ABA) INSENSITIVE5 (ABI5) and
ENHANCED EM LEVEL (EEL), directly activate the expression of major CCGs, including
NYC1 and NYE1 during leaf senescence (94). Other bZIP TFs, ABA-RESPONSIVE ELEMENT
BINDING FACTOR2 (ABF2), ABF3, and ABF4, were also found to promote ABA-mediated
chlorophyll degradation by directly activating the expression of CCGs, such as NYC1, NYE1,
NYE2, and PAO through the ABA-signaling pathways (27). The NAM/ATAF/CUC (NAC) fam-
ily TFs [ANAC016, 019, 046, and 055; ORESARA1 (ORE1)/ANAC092; and RESPONSIVE TO
DESICCATION26 (RD26)/ANAC072], which are positive regulators of leaf senescence, directly
bind to the promoters of a set of major CCGs (83, 88, 93, 94, 135). Moreover, ETHYLENE IN-
SENSITIVE3 (EIN3), a master positive regulator of ethylene signaling, promoted chlorophyll
degradation by physically binding to NYE1, NYC1, and PAO promoters and activating their ex-
pression. The basic helix–loop–helix (bHLH) subgroup IIIe factors MYC2, 3, and 4 directly pro-
mote the expression of NYE1, NYC1, and PAO or indirectly promote expression by mediating
the downstream NAC family TFs ANAC019, 055, and 072, which also activate NYE1, NYE2,
and NYC1 in jasmonic acid (JA) signaling pathways (135). PHYTOCHROME-INTERACTING
FACTOR4 (PIF4) and PIF5 are known to directly regulate NYC1 and/or NYE1 expression (103,
130). More recently, SUPPRESSOR OF OVEREXPRESSION OF CO1, a MADS box family TF,
has been reported as a negative regulator of leaf senescence that directly inhibits the transcription
of the NYC1 and PPH gene (15).
Chloroplast degeneration is accompanied by the progressive loss of proteins and lipids as well as
chlorophyll breakdown. Several senescence-associated stromal and vacuolar proteases accomplish

MYC2
ABF4 ANAC046
ABI5 EEL
PIF5
ABF2 MYC3
ANAC016 ABF3 MYC4
ANAC072 SOC1
PIF4
NYC1 NOL NYE1/SGR1 NYE2 PPH PAO RCCR

Chlorophyll b Chlorophyll a Pheophytin a Pheophorbide a RCC pFCC
HCAR
ANAC019
EIN3
ANAC092/ORE1
Figure 2
Transcriptional regulation of chlorophyll catabolic genes in the pheophorbide a oxygenase (PAO)/phyllobilin pathway. Chlorophyll
catabolic genes are transcriptionally regulated by a variety of family transcription factors. NONYELLOW COLORING 1 (NYC1), which
encodes chlorophyll b reductase for conversion of chlorophyll b into chlorophyll a, is positively regulated by ABSCISIC
ACID–RESPONSIVE ELEMENT BINDING FACTOR 4 (ABF4), ABSCISIC ACID INSENSITIVE 5 (ABI5), ABSCISIC
ACID–RESPONSIVE NAM/ATAF/CUC 019/046/092 (ANAC019/046/092), ENHANCED EM LEVEL (EEL), ETHYLENE
INSENSITIVE 3 (EIN3), MYC2, and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) transcription factors. Another
chlorophyll b reductase gene, NONYELLOW COLORING1-LIKE (NOL), is known to be activated by ANAC092/ORESARA 1 (ORE1).
NONYELLOWINGs (NYEs)/STAYGREENs (SGRs) possessing magnesium-dechelating activity are involved in converting
chlorophyll a to pheophytin a by removing magnesium. ABF2/3/4, ABI5, ANAC016/019/046/072/092, EEL, EIN3, MYC2, and
PIF4/5 transcription factors directly activate expression of the NYE1/SGR1 gene, and ABF2/3/4 and ANAC019/046 activate expression
of the NYE2 gene. Notably, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a MADS box family transcription factor,
directly inhibits the expression of NYE1 and PHEOPHYTINASE (PPH). PPH hydrolyzes pheophytin a to produce pheophorbide a and
phytol. Generation of RCC from pheophorbide a is mediated by PAO, whose expression is positively regulated by ANAC046/092,
ABF4, EIN3, and MYC2/3/4. Red chlorophyll catabolite (RCC) is catalyzed by RCC reductase (RCCR) to generate primary
fluorescent chlorophyll catabolite (pFCC).
key roles in chloroplast protein degradation (39, 85). Lipid-degrading enzymes, such as phospho-
lipase D, lytic acyl hydrolase, and lipoxygenase, also function in the hydrolysis of membrane lipids
Autophagy: an
intracellular
during leaf senescence (109, 110). For example, 13-lipoxygenase that accumulates in the plastid
degradation system by envelope has a role in selective chloroplast degeneration by attacking unsaturated membrane fatty
which unnecessary or acids, which in turn leads to the generation of holes for the mass release of stromal constituents
dysfunctional (104). Furthermore, the importance of lytic vacuoles and autophagy is implicated in chloroplast
cytoplasmic degeneration and recycling (84).
components are
delivered to and
degraded in the
lysosome The Morphological and Metabolic Changes of Mitochondria in Leaf Senescence
The number of mitochondria significantly decreases in senescing Arabidopsis and grapevine leaves
(22, 44, 91). Meanwhile, mitochondria morphology is markedly altered from the elongated,
15.6 Woo et al.

branched structures that are formed by interconnected mitochondria to enlarged, round-shaped

structures during leaf senescence (91). In contrast, mitochondrial integrity and energy status are
maintained until the final stages of leaf senescence in Arabidopsis (22). An integrated transcriptomic
Reactive oxygen
and metabolomic study revealed that mitochondria undergo metabolic alterations to orchestrate species (ROS):
the selective catabolism of both amino acids and fatty acids for optimizing nitrogen redistribution chemically reactive
as leaves senesce (22). molecules and free
Transcriptional coordination between the nucleus and mitochondria appears less tight than radicals derived from
oxygen; examples
that between the nucleus and chloroplasts during leaf senescence (118). Nevertheless, the neces-
include peroxides,
sity of nucleus–mitochondria communication in coordinately regulating gene expression for the superoxide, hydroxyl
proper initiation, execution, and completion of the leaf senescence process is undoubtable. A study radical, singlet oxygen,
of the mitochondrial ATP-dependent protease FtSH4 in Arabidopsis found that the mitochondrial and alpha-oxygen
reactive oxygen species (ROS) functions as a communication signal with the nucleus, which alters
the expression of WRKY TF genes and eventually controls leaf senescence (128). Additional stud-
ies on the detailed mechanisms that underlie the spatiotemporal dynamics of organellar commu-
nication will open up new areas of research to help understand how the organelles communicate
to govern the various biochemical and molecular changes that occur during leaf senescence in
response to continuously changing internal and external environments.
THE FUNCTIONAL AND REGULATORY TRANSITIONS

IN LEAF SENESCENCE
Orderly changes in physiology and biochemistry during leaf senescence are accompanied by
changes in the expression of thousands of senescence-associated genes (SAGs). Recent studies indi-
cate that the dynamic activation of TFs is a key contributor in the regulatory program through
the control of SAG expression in leaf senescence. A process as complex as leaf senescence can
be better understood in the context of molecular regulatory networks; thus, we highlight in this
section the latest understanding of gene regulatory networks (GRNs) involving the NAC and
WRKY TF families, the major TF families that control leaf senescence. Furthermore, this section
describes the recent advent of omics technologies, which have brought remarkable advances to
resolve the complicated processes of leaf senescence.
The Temporal Dynamics and Functions of Gene Regulatory Networks

Involving NAC Transcription Factors
Extensive efforts to define and organize the GRNs involving NAC TFs have elucidated how
GRNs are dynamically regulated to effectively integrate multiple developmental and environ-
mental signals during leaf senescence. For instance, ORE1 is a component of the trifurcate feed-
forward loop for age-induced cell death involving EIN2 and miRNA164 (miR164) (52). EIN3,
which is a key TF in the EIN2-mediated ethylene-signaling cascade, induces the accumula-
tion of the ORE1 transcript by directly repressing miR164 transcription (64). Additionally, five
senescence-associated NAC TFs [NAC-LIKE, ACTIVATED BY AP3/PI (AtNAP)/ANAC029,
ANAC019, ANAC047, ANAC055, and ORE1 SISTER1 (ORS1)/ANAC059] have been identified
as candidate downstream components of EIN2 (48). ORE1 and AtNAP, which are directly acti-
vated by EIN3, regulate both common [ANAC041, 079, and VND-INTERACTING2 (VNI2)]
and different NAC TF targets during leaf senescence. The importance of a GRN involving ORE1
to interweave the light signaling pathway with the leaf senescence program was further inferred
in the multiple feed-forward loops governed by PIF4, PIF5, EIN3, and ABI5 (94). Another co-
herent feed-forward loop involving ORE1 that includes both EIN3 and a few CCGs provides a

molecular basis by which EIN3 and its target, ORE1, accelerate ethylene-mediated chlorophyll
degradation by directly activating CCG expression during leaf senescence in Arabidopsis (88). Thus,
GRNs involving ORE1 function as integrators that coordinate various endogenous and environ-
mental signals during leaf senescence.
In addition to ORE1, other NAC TFs have been implicated in leaf senescence GRNs, along-
side other TF types, upon exposure to diverse stresses. One GRN involving MYB108 and its direct
target, ANAC003, has a regulatory role in dark-stressed leaf senescence (21). Similarly, the GRNs
that involve NAC TFs in Arabidopsis [ANAC032, 072, and 102, and Arabidopsis TRANSCRIP-
TION ACTIVATION FACTOR1 (ATAF1), ATAF2, NAC WITH TRANSMEMBRANE MO-
TIF 1-LIKE 4 (NTL4), and VNI2] and rice (ONAC106, OsNAC2, and OsNAP) regulate leaf
senescence triggered by diverse plant hormones or stresses through modulation of the expression
of SAGs (61, 63, 65, 73, 74, 96, 106, 122).
Systematic analyses have provided valuable information regarding the distinct features and
temporal transition of GRNs involving NAC TFs. A study on the GRNs of ANAC019, 055, and
072 using yeast one-hybrid, time-series gene expression data sets and microarray data analyses il-
lustrated that the expression of each NAC TF gene upon different stress conditions is modulated
by both differential and common upstream TFs (38). Another systematic study on 49 senescence-
associated NACs presented time-dependent networks involving temporal transitions between the
interactions of NAC TFs (49). These time-dependent networks identified a shift from positive
to negative regulation among NAC TFs, which is primarily governed by ANAC017, 082, and
090 before leaf senescence begins. Intriguingly, the shared suppression of senescence-promoting
processes by ANAC017, 082, and 090 included salicylic acid (SA) and ROS responses at prese-
nescent stages. Thus, studies on the time-dependent change of networks reveal unique regulatory
modules, which direct the timely induction of senescence-promoting processes.
The Dynamics and Functions of WRKY Gene Regulatory Networks Underlying

Leaf Senescence
The role of GRN involving WRKY53 in leaf senescence is intensively studied. WRKY53, which
is a positive regulator of leaf senescence, targets various SAGs including pathogen- and stress-
related genes (77). The expression of WRKY53 is directly suppressed by WHIRLY1 (WHY1)
(76) and is at least partially regulated by SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG
2 (SUVH2)-mediated histone modification during leaf senescence (5) (Figure 3). Posttransla-
tional modification also controls the levels of WRKY53; the HECT domain E3 UBIQUITIN
PROTEIN LIGASE5 (UPL5) ubiquitinates and induces the degradation of WRKY53 (79). The
phosphorylation of WRKY53 by MAPK/ERK KINASE KINASE1 (MEKK1) increases its ability
to bind to targets, whereas interactions with EPITHIOSPECIFYING SENESCENCE REGU-
LATOR (ESR) inhibit the DNA-binding activity of WRKY53 (78). A recent study on HISTONE
DEACETYLASE9 (HDA9) unveiled that the HDA9–POWERDRESS (PWR) complex is re-
cruited to W-box-containing promoter regions in a WRKY53-dependent manner that in turn
suppresses the expression of negative senescence regulators (19). Noticeably, WRKY57, which is
one of the downstream targets of the PWR–HDA9–WRKY53 complex and which physically in-
teracts with JASMONATE ZIM-DOMAIN4 (JAZ4) and JAZ8, directly represses the expression
of diverse SAGs in JA-induced leaf senescence (42). Together, these studies provide mechanistic
insights into how a robust regulatory network involving WRKY53 is organized and functions to
modulate leaf senescence. In addition to WRKY53, GRNs involving other WRKY TFs, including
WRKY6, 22, 45, 54, 70, or 75, are thought to hold crucially important roles in leaf senescence (6,
14, 16, 32, 128). Further molecular genetic analyses that combine transcriptome, ChIP-seq, and
15.8 Woo et al.

Chromatin-mediated regulation Ac H3Ac

PWR HDA9
Ac Ac CH3 H3K4me2/3
Ac Ac
WRKY53
SUVH2
CH3
WRKY53 WRKY57 Ac
Transcripts
CH3 CH3
WRKY53 CH3 CH3
WRKY57
Transcripts
Transcriptional regulation
ERF4/8 WRKY57 WRKY57

ESR/ESP
IAA29 JAZ4/8
ESR WRKY53
ESR
WRKY53
WHY1 WRKY57
Target genes
WRKY53
WRKY53 (e.g., SEN1, SAG12)
Target genes
(e.g., WRKY22) Transcripts
Transcripts
Posttranslational regulation
WRKY53 P Phosphorylation
Ub Ubiquitylation
Ub UPL5 MEKK1
Ub
Ub
Ub
Ub
Ub Ub P
WRKY53 WRKY53
WRKY53
degradation
Activation Suppression Change or modification Transcription
Figure 3
Multilayered regulatory mechanisms that involve WRKY53 to control leaf senescence. WRKY53 positively regulates leaf senescence
through diverse means and is also subject to different modes of regulation. (Top) Upregulation of WRKY53 expression during leaf
senescence is at least partially regulated by SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 2 (SUVH2)-mediated histone
methylation. WRKY53 recruits HISTONE DEACETYLASE 9 (HDA9) and POWERDRESS (PWR) to its target sites, which
facilitates the removal of histone acetylation by HDA9 and suppresses WRKY57 expression. (Middle) WRKY57’s interaction with
JASMONATE ZIM-DOMAIN 4 (JAZ4) and JAZ8 represses the expression of diverse senescence-associated genes (SAGs), and its
interaction with indole-3-acetic acid 29 (IAA29) positively regulates jasmonic acid–induced leaf senescence. The expression of
WRKY53 is directly suppressed by WHIRLY 1 (WHY1) during transcriptional regulation. It is also known that the binding activity of
WRKY53 to its target genes can be inhibited by interaction with EPITHIOSPECIFYING SENESCENCE REGULATOR (ESR)
whose transcription is downregulated by ETHYLENE RESPONSE FACTOR 4 (ERF4) and ERF8. (Bottom) Two different
posttranslational modifications control the levels and activities of WRKY53. For instance, UBIQUITIN PROTEIN LIGASE 5
(UPL5) ubiquitinates WRKY53, which induces its degradation, and MAPK/ERK KINASE KINASE 1 (MEKK1) phosphorylates
WRKY53, which increases the binding to its targets.
systems biology analyses will contribute to unraveling the complex GRNs that control leaf senes-
cence, including the identities of GRN components, how GRNs respond to different senescence-
affecting factors, and how these GRNs are interlinked.
Noncoding RNAs
(ncRNAs): functional
RNA molecules that
do not encode proteins Omics Approaches for Temporal Profiling of Molecular Processest
and that regulate gene in Leaf Senescence
expression at the
The advent of transcriptomic technologies has equipped us to address the fundamental principles
transcriptional and
posttranscriptional of leaf senescence on a genome-wide scale in diverse plant species. Transcriptomic studies, partic-
levels ularly in Arabidopsis, have provided multidimensional insights into the functional and regulatory
aspects of leaf senescence. Initial attempts to identify differentially expressed genes during devel-
Degradome
sequencing: opmental leaf senescence in Arabidopsis using a DNA microarray technique for genome-wide pro-
next-generation filing have led to the identification of a distinct chronology of metabolic processes and signaling
sequencing of the 5 pathways during leaf senescence (7, 126). For example, the sequential downregulation of the genes
ends of RNA
involved in anabolic processes, including amino acid biosynthesis, chlorophyll biosynthesis, car-
degradation products,
bon fixation, and photosynthesis, and the sequential upregulation of the genes involved in catabolic
which can reveal many
known and novel plant processes, including autophagy, caspase activity, cell wall degradation, and lipid catabolism, occur
miRNA (small during developmental leaf senescence. The genes involved in hormone signaling pathways, in-
interfering RNA) cluding cytokinin, ABA, and ethylene, are also coordinately altered. Furthermore, the importance
targets of the temporal regulation of NAC and WRKY family TFs for cascades of biological changes is
highlighted in the progression of leaf senescence. Furthermore, a comprehensive, multiple time-
course analysis of Arabidopsis leaf transcriptomes using RNA sequencing (RNA-seq) has enabled
a much deeper understanding of the regulatory features that underlie the leaf senescence pro-
cess, such as temporal coordination of transcriptomes in senescing leaves that is tighter than in
growing ones and importance of the interorganellar coordination between nuclear and chloroplast
transcriptomes (118).
Comparative analyses of diverse leaf senescence transcriptomes provide a deeper understand-
ing of the molecular mechanisms of potential crosstalk among various senescence-promoting fac-
tors (2, 11, 33, 113). Convergent regulatory mechanisms execute leaf senescence processes de-
rived from the different initial response pathways of each senescence-promoting factor. Moreover,
such analyses could facilitate the dissection of complicated molecular programs underlying of leaf
senescence. A recent comparative transcriptome analysis in an ethylene-insensitive mutant and
a constitutive cytokinin response mutant during dark-induced leaf senescence has revealed that
ethylene promotes leaf senescence through the transcriptional activation of the genes involved in
stress-related responses, whereas cytokinin suppresses leaf senescence by maintaining cellular and
translational activities (51).
Another noticeable attempt to delineate the landscape of epigenetic regulation during leaf
senescence is the genome-wide identification and characterization of small noncoding RNAs
(ncRNAs). Genome-wide analyses of the changes in microRNA (miRNA) during leaf develop-
ment and senescence with degradome sequencing revealed that the miRNA–target gene regu-
latory networks that underlie developmental leaf senescence are involved in nutrient mobiliza-
tion and cell structural integrity (107). A systemic study of Argonaute1-enriched small RNAs
(smRNAs) on the expression of SAGs in Arabidopsis and rice identified conserved smRNAs and
target genes in both species, which further indicates their conserved roles in the regulation of
leaf senescence (87). A recent comprehensive smRNA-seq analysis throughout the lifespan of the
Arabidopsis leaf revealed the temporal dynamics and roles of small and long ncRNAs during leaf
senescence (118). An abundance of approximately 30% of smRNAs dynamically alters as leaves
age. An investigation of the age-dependent regulatory networks involving smRNAs and their
15.10 Woo et al.

potential targets further revealed that miRNAs and 21-nucleotide smRNAs regulate distinct pro-
cesses to coordinate transcriptional programs during leaf senescence. Liu et al. (71) identified
168 circular RNAs (circRNAs), including 35 circRNAs that were differentially expressed during
leaf senescence. Construction of the circRNA–miRNA–messenger RNA (mRNA) network during
leaf senescence uncovered that circRNAs are involved in the chlorophyll metabolism and JA sig-
nal transduction through circ-AT5G43822–miR400–PAO and circ-AT3G61420–miR863-3p–JAZ1
networks, respectively; this suggests the participation of circRNAs as new posttranscriptional reg-
ulators for controlling biological processes during leaf senescence. Although epigenomic inves-
tigations of leaf senescence have provided molecular evidence for the importance of diverse epi-
genetic mechanisms that underlie the leaf senescence process, future challenges to characterize a
repertoire of small ncRNAs—such as trans-acting, small-interfering RNAs and small ncRNAs that
originate from ribosomal RNAs (rRNAs), transfer RNAs, or small nuclear RNAs—and long ncR-
NAs will advance our understanding of the ncRNA-involved regulatory mechanisms that modu-
late leaf senescence.

In addition, recent genome-wide analyses of histone modification changes have indicated that
epigenetic regulation is another key regulatory mechanism of leaf senescence. For example, com-
binatorial analyses of chromatin immunoprecipitation sequencing (ChIP-seq) data for two his-
tone modifications and transcriptome data manifested a significant correlation between temporal
changes in gene expression and an active histone mark, trimethylation on histone H3 lysine 4
(H3K4me3), in a subset of genes with altered expression patterns during Arabidopsis leaf senes-
cence. This included WRKY75, which encodes one of the key players of leaf senescence (8, 9).
Such studies have provided insights into the important regulatory role of histone modification in
gene expression during leaf senescence.
To date, proteomic and metabolomic approaches to leaf senescence research still lag behind
DNA- and RNA-based high-throughput techniques, although they provide a global physiochem-
ical view of cellular status and its regulation during leaf senescence. Further investigations that
combine proteomic approaches and the immunoprecipitation of protein complexes are required
to unmask novel regulatory aspects of leaf senescence programs.
MULTILAYERED REGULATION OF STRESS-INDUCED LEAF

SENESCENCE
Leaf senescence proceeds with age and involves intricate regulatory programs that respond to
various external factors and internal factors, including all of the leaf’s developmental processes
(Figure 1). This section highlights key findings that illustrate the complexity of the regulatory
programs of stress-induced leaf senescence, which are finely regulated at multiple chromatin, tran-
scriptional, posttranscriptional, and posttranslational regulatory levels.
The Chromatin-Mediated Regulation of Stress-Induced Leaf Senescence

The control of chromatin conformation through histone modification and chromatin-remodeling
enzymes is an emerging key regulatory mechanism of leaf senescence that can be triggered by
either biotic or abiotic stresses. A few recent reports have provided further evidence of the piv-
otal roles of histone modification in the regulation of stress-induced leaf senescence. A defi-
cient mutant of the Arabidopsis HDA9 gene exhibits delayed dark-induced leaf senescence phe-
notypes (19). WRKY53 recruits HDA9 and PWR to its target sites, which facilities the removal
of Histone 3 acetylation (H3Ac) by HDA9 and suppresses the expression of key negative regula-
tors of senescence, such as AUTOPHAGY9 (ATG9) and WRKY57 (Figure 3). A loss-of-function

mutant of an Arabidopsis histone acetyltransferase gene, HOOKLESS1 (HLS1), leads to enhanced

leaf senescence phenotypes by dark, pathogen, or ABA treatment (66). HLS1 enhances the
expression of WRKY33 and ABI5—which, respectively, are a well-known pathogen and an ABA-
signaling regulator—through modulating H3Ac on chromatin. The significance of the chromatin-
mediated regulation in dark-induced leaf senescence has also been highlighted by characteriz-
ing the mutations of chromatin-modifier mutations, such as DEFECTIVE IN RNA–DIRECTED
DNA METHYLATION1 (DRD1) and DECREASED DNA METHYLATION1 (DDM1) (20). Fur-
ther studies are needed to uncover both how diverse chromatin modifiers differentially re-
spond to various internal and external factors to effectively regulate leaf senescence and how
TFs and chromatin modifiers cooperate to regulate gene expression during stress-induced leaf
senescence.
The Transcriptional Regulation of Stress-Induced Leaf Senescence

This section summarizes the progress made toward the identification and characterization of TFs
that have a role in the regulation of stress-induced leaf senescence and describes how such present
knowledge lays a foundation for addressing important challenges in unveiling the TF-mediated
regulatory mechanisms of stress-induced leaf senescence.
The NAC TF family is the most intensively characterized TF family involved in stress-induced
leaf senescence in diverse plants. Studies of Arabidopsis have provided evidence that NAC TFs
mediate the regulation of leaf senescence by coupling stress-related signaling with senescence-
related transcriptional modules. For example, ANAC016, a positive regulator of dark, salt, and
oxidative stress–induced leaf senescence, directly binds to promoters of AtNAP and ORS1 and
enhances their transcript levels (54). Moreover, both ANAC016 and AtNAP directly bind to
the promoter of ABF2 that encodes a TF that is important in the ABA signaling pathway and
represses its transcription (95). Recently, SGR1/NYE1, which is a major CCG, was identified as
another direct downstream target of ANAC016 (93). Another NAC TF that functions as a posi-
tive regulator of dark and oxidative stress–induced leaf senescence, ATAF1, activates ORE1 tran-
scription and represses GOLDEN2-LIKE1 (GLK1) transcription by directly binding to their pro-
moters (28). Additionally, ANAC032 is a positive regulator of dark, oxidative, osmotic, and salt
stress–induced leaf senescence by modulating the expression of SAGs, such as NYE1, SAG113, and
SAG201 (73). RD26, a positive-regulator of dark-induced leaf senescence (63), directly enhances
the expression of diverse repertoires of downstream genes, including CHLOROPLAST VESIC-
ULATION, which is involved in chloroplast protein degradation; LYSINE KETOGLUTARATE
REDUCTASE/SACCHAROPINE DEHYDROGENASE, which is involved in lysine catabolism;
and ALPHA-AMYLASE1, SUGAR-PORTER FAMILY PROTEIN1, and SWEET15, which are in-
volved in carbohydrate metabolism and transport (43).
The involvement of the APETALA 2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) TF
family in regulating stress-induced leaf senescence has also been thoroughly studied. AtERF4 and
AtERF8 have positive roles in dark-induced leaf senescence via direct downregulation of ESR),
which negatively regulates WRKY53 (56) (Figure 3). In addition, an AP2/ERF family TF in
mulberry, MnDREB4A, is known as a negative regulator of leaf senescence triggered by diverse
stresses, including drought and salt stresses (72). Overall, the intensive studies over the past sev-
eral years have clearly illuminated the significance of TF-mediated regulation as a key regula-
tory mechanism underlying stress-induced leaf senescence. Further systematic studies designed
to reveal the characteristics and dynamics of protein–protein and/or protein–DNA interactome
networks involving numerous TFs will be necessary for more comprehensive insights into global
TF-mediated regulatory mechanisms of stress-induced leaf senescence.
15.12 Woo et al.

Posttranscriptional Regulation of Stress-Induced Leaf Senescence

On the basis of recent genome-wide analyses of ncRNAs during leaf senescence (87, 107, 118) and
the well-characterized miRNA-mediated regulatory networks involving miR164 or miR319 (52,
98), the contributions of posttranscriptional regulation to leaf senescence are evident. This section
discusses regulations mediated by RNA splicing and processing, where advances in posttranscrip-
tional regulatory mechanisms of stress-induced leaf senescence are recently emerging. Discovery
of the roles of Arabidopsis minor spliceosomal protein U11–48K as a positive regulator in dark-
induced leaf senescence is the first example of the involvement of RNA splicing in the regulation
of stress-induced leaf senescence (120). U11-48K is one of the ribonucleoproteins unique to the
minor spliceosome that catalyzes the splicing of minor U12-type introns. The positive correla-
tion between the severity of abnormal developmental phenotypes and the degree of impairment
in U12 intron–splicing in the u11–48k mutants further indicates that correct splicing of U12 in-
trons is necessary for normal plant growth and development, including dark stress–induced leaf
senescence. A more recent study on the sugarcane MYB TF ScMYB2 provides elegant evidence
supporting that differential expression of alternatively spliced transcripts might be an important

posttranscriptional regulatory mechanism in the control of drought stress as well as leaf senes-
cence (31). Likewise, the importance of RNA processing in stress-induced leaf senescence was
initially demonstrated in a study of the chloroplast RNA splicing and ribosome maturation family
member subfamily 4 (CFM4) in Arabidopsis. The cfm4 mutant exhibited delayed leaf senescence
phenotypes and abnormal patterns of chloroplast rRNA processing (60), indicating the impor-
tance of rRNA processing in stress response, including dark-induced leaf senescence. The potato
(Solanum tuberosum) RNA-binding protein StUBA2a/b, when overexpressed in Arabidopsis, causes
early leaf senescence phenotypes under dark-stress conditions, which is accompanied by altered
expression of SA biosynthetic and signaling genes as well as autophagy-associated genes (81). De-
spite the importance of posttranscriptional regulation in biological processes, there are still rela-
tively few examples for stress-induced leaf senescence. Thus, the next key step will be to investigate
the roles of posttranscriptional regulatory mechanisms such as mRNA processing (5 -capping and
3 -end processing), mRNA modification, and mRNA export machineries in stress-induced leaf
senescence.
Posttranslational Regulation of Stress-Induced Leaf Senescence

Another regulatory layer of stress-induced leaf senescence involves diverse posttranslational modi-
fications, which can influence the conformation, activity, stability, and localization of proteins. One
of the most extensively characterized posttranslational modification mechanisms in the regula-
tion of stress-induced leaf senescence is protein phosphorylation and dephosphorylation through
kinases and phosphatases. For example, an S-domain receptor-like kinase, OsSIK2, is a negative
regulator of leaf senescence, which is triggered by diverse environmental stresses such as darkness,
drought, and salt (17). Another rice receptor-like cytoplasmic kinase, Oryza sativa BILATERAL
BLADE SENESCENCE1 (OsBBS1)/OsRLCK109, is shown to function as a negative regulator
of dark- or salt-induced leaf senescence (125).
Recent studies on the SNF1-RELATED KINASE (SnRK) genes in Arabidopsis have further
revealed the importance of protein phosphorylation–mediated regulation of stress-induced leaf
senescence. ABA-activated SnRK2s phosphorylate ABFs and RELATED TO ABA INSENSI-
TIVE3/VP1 (RAV1) TFs, which subsequently activates the expression of SAGs including ORE1.
By contrast, another Arabidopsis SnRK, SnRK1/Arabidopsis SNF1 KINASE HOMOLOG 10
(AKIN10) is a negative regulator of dark-induced leaf senescence (45). SnRK1/AKIN10
directly interacts, phosphorylates, and destabilizes EIN3, which suppresses expression of EIN3

target genes including ORE1. Additional evidence on the importance of reversible protein
phosphorylation in stress-induced leaf senescence is supported by the characterization of the Ara-
bidopsis SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP) gene. SSPP negatively
regulates dark-induced leaf senescence through direct dephosphorylation of SENESCENCE-
ASSOCIATED RECEPTOR-LIKE KINASE (SARK), a positive regulator of leaf senescence
(119).
Protein ubiquitylation is another well-defined posttranslational modification in the regula-
tion of stress-induced leaf senescence. Two plant U-box (PUB) E3 ubiquitin ligases, PUB12 and
13, are involved in defense response and dark stress–induced leaf senescence (134). Similarly,
PUB44/SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1)/NOT ORE-
SARA 1 (NORE1) have also been reported to mediate signals from temperature- and humidity-
dependent defense programs and leaf senescence (23, 59, 115). A recent study demonstrated that
the ubiquitin receptor DA1 and the E3 ubiquitin ligase BIG BROTHER (BB) in Arabidopsis, both
of which are known to restrict leaf size, also have roles as positive regulators of dark-induced
leaf senescence (114). Further systemic studies to identify novel posttranslational modifiers that
function in this process and to understand the regulatory networks involving upstream regulators,
downstream targets, and interaction partners will help to unravel the posttranslational regulatory
mechanisms of stress-induced leaf senescence.
CROSSTALK BETWEEN HORMONE SIGNALING

AND LEAF SENESCENCE
Leaf senescence of higher plants involves genetically and environmentally regulated processes and
intimate crosstalk with hormonal interactions (26, 68). Indeed, global gene expression analysis and
characterization of genetic mutants have revealed that all classical plant hormones can potentially
affect leaf senescence at all stages of leaf development. Recently, regulatory molecules, including
TFs that are associated with hormone signaling, have been identified as key regulators of leaf
senescence (Figure 4).
JA, a lipid-derived plant hormone ubiquitous in the plant kingdom, regulates diverse plant de-
fense responses and various development processes (41). The effects of JA on leaf senescence have
long been known, based on findings that the endogenous JA level increases in senescent leaves
and that exogenous application of JAs induces leaf senescence in a variety of plant species. Con-
sistent with the increased JA level during leaf senescence, expression of genes involved in the JA
biosynthesis pathways LIPOXYGENASE1 (LOX1), LOX3, LOX4, ALLENE OXIDE SYNTHASE
(AOS), and ALLENE OXIDE CYCLASE1 (AOC1), is increased in senescing leaves (37). The mu-
tant of CORONATINE INSENSITIVE1 (COI1), a component of a receptor for JA, abolishes
JA-induced leaf senescence, indicating that key components of JA signaling pathway also have
roles in leaf senescence (37).
JAZ4 and JAZ8, key repressors in JA signaling pathway (108), were found to physically inter-
act with WRKY57 to negatively regulate JA-induced leaf senescence (42). WRKY57 further in-
teracted with the INDOLE-3-ACETIC ACID INDUCIBLE29 (IAA29) protein, a repressor of
auxin signaling pathways, which positively regulates JA-induced leaf senescence, suggesting that
JA and auxin antagonistically regulate JA-induced leaf senescence through WRKY57. Another
JAZ protein, JAZ7, controlled dark-induced leaf senescence (124).
Recently, MYC2, 3, and 4, targets of JAZ repressors, were identified as functioning redun-
dantly to activate JA-induced leaf senescence by binding to and activating the promoter of SAG29
(86). Interestingly, bHLH subgroup IIId factors, bHLH03, 13, 14, and 17, also bind to the
SAG29 promoter and repress its expression to attenuate MYC2/3/4-activated JA-induced leaf
15.14 Woo et al.

Auxin SA Cytokinin
Young
Ub
ORE12/AHK3 Ub
Ub Ub ARR2 P
AHPx P
YUC6 ARF2 IAA29 ANAC090 ICS1 Nucleus
ARR2
ICS1 CRF6 CRF6

CRFs
ANT SA-response genes (PR1) Cytokinin-response genes

Ethylene Light/dark
Dark
Old
IAA29
PIF4/5
EIN2 AtNAP/ANCA029
JA
AtNAP/ANAC029
ABI5/EEL
EIN3
Dark COI1 AAO3

ORE1/ANAC092 ABA AAO3
ORE1/ANAC092
JAZ7 JAZ4/8 WRKY57

ATAF1
JAZ7 AtNAP/ANCA029
AtNAP/ANAC029
NCED3, ABCG40
SEN4/SAG12
MYC2/3/4 NCED3, ABCG40
PYL9
NACs (VNI2, ANAC041/079)
ANAC019/055/072
ABF2/3/4
bHLH03/13/14/17
SAGs (BFN1, SAG29, SINA1)
SAG12
SAG29 CCGs (NYE1, NYC1, PAO)

GA WRKY6 SA
WRKY45 WRKY75 WRKY46/51/63

SID2
SAG12/SAG113 SAG13/SGR CAT2 SID2
Activation Suppression Transcription

(Caption appears on following page)

Figure 4 (Figure appears on preceding page)

Crosstalk between hormone signaling and leaf senescence: effects of hormones on leaf senescence. (Top) In young leaves, auxin and
cytokinins act as major antisenescing hormones. Auxin suppresses the expression of SAGs through the ARF2–ANT pathways. ARR2
and CRF6 are downstream components in the pathway by which AHK3 and TCS mediate enhanced leaf longevity in response to
cytokinin. (Bottom) In old leaves, JA, ethylene, ABA, SA, and GA signaling pathways initiate and promote leaf senescence. MYC2/3/4,
targets of JAZ repressors in JA signaling pathways, activate CCGs and SAGs in dark-triggered and JA-triggered leaf senescence.
ANAC019/055/072, downstream NAC TF genes of MYC2/3/4, also activate CCGs for chlorophyll degradation during leaf senescence.
ORE1/ANAC092 involves feed-forward loops integrating ethylene (EIN2 and EIN3) and ABA (ATAF1 and ABI5/EEL) into the
developmental aging program. ORE1 and AtNAP/ANAC029 activate NACs, CCGs, and SAGs to promote leaf senescence. The key
regulators of ABA signaling pathways ABF2/3/4 also directly activate CCGs and SAGs through the PYLs–PP2C–SnRK2 regulatory
module for ABA-triggered leaf senescence. Besides NAC TFs, WRKY TFs act as positive regulators of leaf senescence.
WRKY46/51/63/75 promote leaf senescence through the production of endogenous SA, whereas WRKY6/45 involve GA-triggered
leaf senescence by activating CCGs and SAGs. WRKY75 further represses the expression of CAT2, indicating a tripartite amplification
loop involving WRKY75, SA, and ROS. Abbreviations: AAO, ABSCISIC ALDEHYDE OXIDASE; ABA, abscisic acid; ABC,
ATP-BINDING CASSETTE; ABF, ABSCISIC ACID–RESPONSIVE ELEMENT BINDING FACTOR; ABI, ABSCISIC ACID
INSENSITIVE; AHK, ARABIDOPSIS HISTIDINE KINASE; ANAC, ABSCISIC ACID–RESPONSIVE NAM/ATAF/CUC; ANT,
AINTEGUMENTA; ARF, AUXIN RESPONSE FACTOR; ARR, ARABIDOPSIS RESPONSE REGULATOR; ATAF,
ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR; AtNAP, NAC-LIKE, ACTIVATED BY AP3/PI; BFN,

BIFUNCTIONAL NUCLEASE; bHLH, basic helix–loop–helix; CAT, CATALASE; CCG, chlorophyll catabolic gene; COI,
CORONATINE INSENSITIVE; CRF, CYTOKININ RESPONSE FACTOR; EEL, ENHANCED EM LEVEL; EIN,
ETHYLENE INSENSITIVE; GA, gibberellin; IAA, indole-3-acetic acid; JA, jasmonic acid; JAZ, JASMONATE ZIM-DOMAIN;
NAC, NAM/ATAF/CUC; NCED, NINE-CIS-EPOXYCAROTENOID DIOXYGENASE; NYC, NONYELLOW COLORING; NYE,
NONYELLOWING; ORE, ORESARA; PIF, PHYTOCHROME-INTERACTING FACTOR; PP2C, PROTEIN PHOSPHATASE
2C; PYL, PYRABACTIN RESISTANCE 1–LIKE; ROS, reactive oxygen species; SA, salicylic acid; SAG, senescence-associated gene;
SEN, SENESCENCE; SGR, STAYGREEN; SID, SALICYLIC ACID INDUCTION–DEFICIENT 2; SINA, SEVEN IN ABSENTIA;
SnRK, SNF1-RELATED PROTEIN KINASE; TCS, two-component system; TF, transcription factor; VNI, VND-INTERACTING.
senescence. This antagonistic regulation by activators and repressors would mediate JA-induced
leaf senescence at levels suitable for plant survival in fluctuating environmental conditions.
Additionally, MYC2/3/4 proteins were found to regulate JA-induced chlorophyll degradation
directly by binding to the promoters of major CCGs. Furthermore, JA signaling downstream
of the MYCs ANAC019/055/072 also regulates the expression of a similar set of CCGs (135),
indicating the hierarchical and coordinated regulation of JA-induced chlorophyll degradation by
two TF family genes.
ABA is one of the most effective plant hormones in terms of promoting leaf senescence. Ex-
ogenous application of ABA induces senescence-associated mRNAs and promotes leaf senescence
(58), indicating the presence of a link between ABA signaling and leaf senescence. Moreover, a va-
riety of biotic and abiotic stresses elevate ABA level and activate ABA-signaling pathways leading
to senescence (92).
Recent studies on ABA-signaling pathways revealed that three essential core components—
PYRABACTIN RESISTANCE 1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPO-
NENTS OF ABA RECEPTOR (RCAR), clade A type 2C PROTEIN PHOSPHATASES
Osmotic potential: (PP2Cs), and subclass III SnRK2s—mediate ABA-promoted leaf yellowing and senescence. A
a measure of the large-scale screening of transgenic plants overexpressing the PYL family showed that PYL9 pro-
tendency of a solution
motes drought resistance and leaf senescence by inhibiting PP2Cs and activating SnRK2s (133).
to withdraw water
from pure water by Intriguingly, under drought conditions, leaf senescence apparently helps to generate a greater os-
osmosis across a motic potential gradient, which causes water to preferentially flow to developing tissues for plant
differentially survival. This observation is particularly notable in that leaf senescence is a critical strategy to
permeable membrane survive under extreme drought conditions and in that a tight link between drought survival and
leaf senescence exists through ABA signaling.
Recently, ABF2/3/4 were found to activate the expression of SAG29 (and possibly other SAGs,
such as SAG12) and CCGs through the core system (PYLs–PP2C–SnRK2) of ABA-signaling
15.16 Woo et al.

pathways. This suggests roles for ABF2/3/4 in the regulation of ABA-triggered leaf senescence
and chlorophyll degradation (27). Several NAC TFs are also involved in chlorophyll degradation
via induction of the ABA biosynthetic genes. AtNAP increases ABA levels and induces the ex-
pression of genes involved in chlorophyll degradation (121). Notably, the rice NAC TF OsNAC2
promotes leaf senescence by inducing ABA biosynthetic genes and downregulating ABA catabolic
genes. OsNAC2 also directly regulates the chlorophyll degradation genes OsSGR and OsNYC3
(74). Collectively, the enhanced levels of ABA through the induction of ABA biosynthetic genes
support the role of ABA pathways in leaf yellowing and senescence.
SA has long been known to promote natural leaf senescence (68, 90). The endogenous SA
gradually increases as a leaf ages, which induces the expression of several SAGs during leaf
senescence. More than 70% of the WRKY gene family members belonging to different groups
are responsive to pathogen infection and SA treatment, and a large portion of them are im-
plicated as regulators of senescence in Arabidopsis. WRKY53 and WRKY70 have roles as posi-
tive and negative regulators of senescence (6, 77), respectively, and mutant studies on WRKY54,
the closest homolog of WRKY70, indicated the functional redundancy or cooperative role of
WRKY54 and WRKY70 in leaf senescence (6). Furthermore, WRKY46, WRKY51, WKRY63,
and WKRY75 promote SA production by directly inducing the transcription of SALICYLIC
ACID INDUCTION–DEFICIENT 2 (SID2) (128). WRKY75 further repressed ROS scavenging
by repressing CATALASE 2 (CAT2) (32), indicating that a tripartite amplification loop involving
WRKY75, SA, and ROS has a major role in promoting leaf senescence.
ANAC090 was recently identified as a negative regulator of SA-mediated leaf senescence (49);
loss of ANAC090 leads to increased SA levels and accelerated leaf senescence. In general, SA and
ROS mutually promote each other’s accumulation during leaf senescence; however, anac090 mu-
tants show no significant difference in the levels of superoxide and H2 O2 from the wild type.
Crosstalk between SA and ROS responses can maintain the levels of antioxidants sufficiently to
protect the senescing leaf cells from premature death, ensuring the slow degeneration of cells
during leaf senescence.
The gaseous phytohormone ethylene is a widely acknowledged positive regulator of leaf senes-
cence. Exogenous ethylene treatment accelerates leaf senescence, and the ethylene biosynthetic
genes 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE (ACS) and ACC
OXIDASE (ACO) are upregulated in senescing leaves, consistent with an increased ethylene level
during leaf senescence (113). Leaf senescence progression is fine-tuned by regulating the sta-
bility of key proteins in ethylene-signaling pathways to balance growth and senescence during
leaf development. For example, expression of ACS7 is upregulated during both natural and dark-
induced leaf senescence; and high accumulation of ACS7 protein leads to precocious leaf senes-
cence (105). Intriguingly, degradation of ACS7 depends on the first 14 N-terminal amino acid
residues, and the degradation of ACS7 is negatively regulated by leaf senescence signaling, allow-
ing ethylene to reach an appropriate level for leaf development. In addition, the protein levels of
EIN3 are also modulated by an evolutionarily conserved cellular energy sensor, SnRK1/AKIN10
(45). SnRK1/AKIN10 blocks the photosystem II (PSII) electron transport chain leading to intra-
cellular sugar starvation/energy deprivation. Thus, SnRK1/AKIN10 activity enhances cell viabil-
ity and delays organ senescence under energy restricted conditions.
The roles of gibberellins (GAs), a large group of tetracyclic diterpenoids, in leaf senescence
have been elusive, particularly due to their involvement in diverse developmental processes (1).
However, studies suggest that GAs are also involved in the senescence process. Leaf senescence
is retarded in the ga1–3 mutant, where GA biosynthesis is blocked and DELLA proteins GA
INSENSITIVE (GAI), REPRESSOR OF GA1-3 (RGA), RGA-LIKE1 (RGL1), RGL2, and
RGL3—the negative regulators of GA signaling pathways—abnormally accumulate (18). In

contrast, despite the deficiency in GA biosynthesis, leaf senescence occurred earlier in the ga1-3
gai-t6 rga-t2 rgl1-1 rgl2-1 mutant (abbreviated as Q-DELLA/ga1-3), suggesting that DELLA
repression retards natural leaf senescence. Recently, WRKY45 was found to function in the
Two-component
system (TCS): GA-mediated signaling pathway to positively regulate age-triggered leaf senescence (16). Both
a stimulus-response the delayed leaf senescence phenotype in the loss-of-function mutant wrky45 and the accelerated
coupling mechanism leaf senescence phenotype in WRKY45-overexpressing plants establish WRKY45 as a positive
to sense and respond regulator of leaf senescence. WRKY45 interacts with the DELLA protein RGL1, and this
to different
interaction impairs the transcriptional activities of WRKY45 on target genes, including SAG12
environments; a
membrane-bound and SAG113. Similarly, the DELLA protein RGA negatively regulates dark-induced senescence
sensor kinase and a and chlorophyll degradation through interaction with WRKY6 (131). The interaction results
DNA-binding in impaired transcriptional activation by WRKY6 of the downstream target genes, including
response regulator SAG13 and SGR. Under darkness, WRKY6 expression increases, but the expression of DELLAs
decreases, and together these attenuate the repression of RGA on WRKY6 transcriptional
activity. Together, the GA–WRKY link observed in these studies provides new insight into the
transcriptional regulation of GA-promoted leaf senescence in Arabidopsis.

It is well known that the exogenous application of active cytokinins and an increase in the
content of endogenous cytokinins can delay senescence (68). The senescence-delaying effect of
cytokinin depends upon the canonical two-component system (TCS) in Arabidopsis. Perception of
cytokinin by the receptor kinase ARABIDOPSIS HISTIDINE KINASE3 (AHK3) and subsequent
phosphorylation of the type B response regulator ARABIDOPSIS RESPONSE REGULATOR2
(ARR2) are required for increased leaf longevity (50). CYTOKININ RESPONSE FACTORs
(CRFs), a subset of the plant-specific AP2/ERF domain–containing TF family, have been shown
to act as a side branch of the canonical TCS pathway. CRF6 was reported to act as a negative
regulator of dark-induced senescence (137). A recent study further demonstrated that expression
of CRF6 is induced by oxidative stress as well as by cytokinin and that CRF6 functions as a negative
regulator to repress cytokinin-associated genes during oxidative stress (136), which indicates that
CRF6 attenuates the cytokinin signaling to allow an improved stress response.
Early studies indicate that auxins have a role in the suppression of leaf senescence (82, 101).
An increased level of auxin in YUCCA 6 (YUC6)-overexpressing plants delayed leaf senescence
in natural and dark-induced senescence conditions by reducing SAG12 expression (53). The
thiol-reductase activity of YUC6 further mediates a delay in leaf senescence via the activation of
genes involved in redox signaling and auxin redistribution (13). AUXIN RESPONSE FACTOR2
(ARF2), a repressor of auxin signaling, was found to function in the auxin-mediated regulation of
leaf longevity by suppressing the expression of SAGs (69). Recently, AINTEGUMENTA (ANT),
a member of the AP2/ERF TF family, was identified as a downstream regulator of ARF2 (24). The
loss-of-function ant-1 mutant shows premature leaf senescence, whereas overexpression of ANT
leads to a delay in leaf senescence. Moreover, ant-1 mutant represses the delayed leaf senescence
phenotype in arf2-5 mutant plants. Taken together, these findings indicate that ANT is involved
in the regulation of leaf senescence downstream of ARF2.
OTHER EMERGING REGULATORY MECHANISMS OF LEAF

SENESCENCE
This section highlights other regulatory mechanisms of leaf senescence.
Light Signaling and Leaf Senescence

Light is arguably the most significant environmental factor and light signaling pathways have an
important role in regulating leaf senescence. Phytochromes are photoreceptors that signal red
15.18 Woo et al.

and far-red light information. In Arabidopsis, red light negatively regulates leaf senescence, while
far-red light positively regulates it (67). Phytochrome signaling is mediated through various PIFs
(12). Positive roles of PIF1, 3, 4, and 5 in the control of age- and dark-induced leaf senescence
Circadian clock
have been well demonstrated (94, 103, 130). Upon prolonged darkness, increased PIF activities oscillator:
directly enhance the expression of multiple targets of senescence regulatory components, and a biochemical
this in turn triggers leaf senescence (94). PIF4 and PIF5 regulate dark-induced leaf senescence, oscillator that cycles
at least partially through direct activation of ABI5, EEL, and EIN3. It was further revealed that with a stable phase and
is synchronized with
ORE1 is a direct target of PIF4, PIF5, and their three targets, ABI5, EEL, and EIN3, indicat-
solar time
ing that ORE1 functions as a mediator to integrate phytochrome B–mediated light signaling to
promote leaf senescence in light-deprived conditions. Moreover, PIF4 directly activates the ex- Evening complex
(EC): a critical
pression of the chlorophyll-degeneration regulatory gene NYE1 and also directly represses GLK2,
component of the core
a G2-LIKE TF gene that is important for maintenance of chloroplast activity (103, 130). This oscillator in regulating
finding is in line with the previous observation that ORE1 sequesters GLK1 and GLK2 through
circadian outputs;
protein–protein interaction, which leads to a reduction in the transcriptional activity of GLKs as mutation of any EC
component leads to
leaves get older (89). It is thus now obvious that the PIF proteins are core components that trans-
circadian arrhythmia
duce photoreceptor-mediated light signaling to key leaf senescence regulators. A recent report
reveals that phytochrome A and phytochrome B antagonistically regulate far-red-light-enhanced
leaf senescence, which is mediated by WRKY6 (67). Further studies are certainly required to elu-
cidate the detailed molecular mechanisms of how light signaling pathways are interconnected with
other internal and external senescence-regulating factors.
Circadian Rhythm, Aging Clock, and Leaf Senescence

Temporal regulation of development is critical for organisms’ fitness, so mechanisms measuring
the passage of time and executing developmental transitions at the appropriate time should exist.
However, there is still no clear understanding of how plants recognize the passage of time.
The circadian clock is a part of the endogenous time-keeping mechanisms that allow organisms
to anticipate and prepare for daily and seasonal changes in their surrounding environments (75)
and is a good candidate for temporal coordination. Indeed, there is accumulating evidence sup-
porting that the circadian clock is linked with leaf senescence through cross-regulatory networks.
Kim et al. (47) showed that the circadian clock is affected by leaf age. The circadian period gets
shorter in older leaves, which are regulated through the circadian clock oscillator TIMING OF
CAB EXPRESSION1 (TOC1). This finding provides the first glimpse into understanding how
age-dependent changes in the circadian clock are incorporated into age-dependent developmental
decisions.
The Arabidopsis circadian evening complex (EC), composed of EARLY FLOWERING 3
(ELF3), ELF4, and LUX ARRHYTHMO (LUX), was shown to negatively regulate JA-induced
leaf senescence (132). All mutations in the EC components showed accelerated natural leaf senes-
cence and more pronounced JA-induced leaf senescence, whereas plants with an overexpression
of ELF3 displayed the delayed senescence symptom when leaf senescence was artificially induced
by JA treatment. The EC component LUX directly binds to the promoter of the MYC2 TF gene,
which encodes a key activator of JA-induced leaf senescence, thereby leading to repression of
MYC2 expression. Furthermore, the myc2 myc3 myc4 triple mutation abolished the accelerated JA-
induced leaf senescence seen in EC mutants, confirming that a core component of the circadian
clock gates JA signaling via MYC TFs to regulate leaf senescence.
CIRCADIAN CLOCK–ASSOCIATED 1 (CCA1)-mediated control of leaf senescence was
also evident (102, 117). Mutation of CCA1 causes early leaf senescence, suggesting that there is
a negative role for CCA in the regulation of leaf senescence. Intriguingly, CCA1 represses the

positive senescence regulator ORE1 and activates the chloroplast maintenance gene GLK2 by
directly binding to the promoters of both genes. It is thus conceivable that CCA regulates the
expression of ORE1 and GLK2 to inhibit leaf senescence at the juvenile stage, but with aging,
Nitrogen
remobilization the declined expression of CCA1 and GLK2 attenuates the inhibition of leaf senescence, and the
efficiency: the accumulation of ORE1 promotes senescence initiation.
proportion of nitrogen It is interesting that ORE1 is also under circadian regulation. This implies that ORE1 is an
in the crop or crop integrator of age-dependent senescence and the circadian clock. A recent report revealed that
component at anthesis
PSEUDO-RESPONSE REGULATOR9 (PRR9), a component of the circadian clock, regulates
that is not present at
harvest leaf senescence by directly enhancing expression of ORE1 and also by repressing miR164 expres-
sion, setting a feed-forward loop (46). Delineation of the interaction networks between the cir-
cadian clock and leaf senescence systems will contribute to future understanding of how plant
developmental processes are controlled in aging.
Age Gating: Function of a Senescence Regulatory Gene

The circadian system shows circadian gating: The same degree of input shows a different degree of
output depending on the time of day (29, 55). The RECEPTOR PROTEIN KINASE 1 (RPK1) gene
leads to very different phenotypic effects on leaf development: Arabidopsis leaves show enhanced
senescence when RPK1 is induced in old leaves, whereas the leaves show arrested growth with
no sign of senescence when RPK1 is induced in young leaves (58). Thus, the RPK1 gene leads
to drastically different phenotypes depending on leaf age, indicating that an age gating of gene
function may exist in plants.
AGRONOMIC IMPLICATION OF LEAF SENESCENCE RESEARCH

Recent advances in physiological and molecular understandings of leaf senescence have led to
various strategies of manipulating leaf senescence for agricultural improvement. Here, we discuss
two major approaches: One is to identify genes or develop strategies for the improvement of
nitrogen remobilization efficiency, and the other is to manipulate key senescence regulatory genes.
Development of Strategies for Efficient Nitrogen Utilization

Nitrogen is the element predominantly remobilized during leaf senescence. Recent evidence
shows that autophagy is also an essential process for nitrogen remobilization from leaves to seeds
(4, 36). A nitrogen-15 tracing study of autophagy mutants defective in autophagosome expan-
sion and enclosure—atg5, atg9, and atg18 in Arabidopsis—revealed that the autophagy machinery
participates in the control of up to 60% of nitrogen remobilization to seeds under a low-nitrate
condition and up to 20% under a high-nitrate condition (30). Due to their decreased nitrogen-
remobilization capacity, these atg mutants display lower seed production regardless of plant nutri-
tion and overaccumulate nitrogen compounds, such as soluble proteins, ammonium, and peptides
identified as degradation products of the Rubisco in rosette leaves, during senescence. The role of
autophagy in Rubisco degradation and nitrogen remobilization was also confirmed in the rice Os-
atg7 mutant and the maize atg12 mutant (62, 116). Conversely, ATG5- and ATG7-overexpressing
Arabidopsis exhibit delayed senescence and enhanced growth, seed set, and seed oil content (80),
providing further mechanistic insight into genetic stimulation of autophagy for the improvement
of plant productivity.
Additional investigation of the complex regulatory mechanisms of autophagy is still needed,
especially in view of utilizing autophagy for agricultural purposes. It should be noted that au-
tophagy rather than just the proteolysis process itself might control protein degradation as a
15.20 Woo et al.

shuttle between the substrates and the protease. Thus, the identification of autophagy cargos
and the respective proteases that contribute to nitrogen use efficiency will also be of immense
importance.
Nitrogen use
Chloroplast degradation during leaf senescence generates nutrients, such as sugars, lipids, efficiency: the ratio
and amino acids for remobilization. It is also well known that glutamine and asparagine, which between the amount of
have two nitrogen atoms per molecule, are preferred molecules to transport nitrogen exported in nitrogen removed
the phloem sap of plants. Overexpression of the cytosolic GLUTAMINE SYNTHETASE1 (GS1), from the field by crops
and the amount of
which functions in the metabolism of nitrogen by catalyzing the condensation of glutamate and
nitrogen fertilizer
ammonia to form glutamine, has been successful in numerous cases with the goal of improving applied
crops’ nitrogen use efficiency (47). However, the outcome has generally been inconsistent, pos-
Phloem loading: the
sibly due to deregulation of GS1 activity via metabolic imbalances (10, 92). Thus, spatially and
process of loading
temporally regulated GS overexpression strategies should be considered. carbon into the
In contrast to the numerous studies on the transgenic modification of GS1 (111), very few
phloem sieve tubes at

studies have been carried out on the effects of plastidic GS2. Wheat varieties with the TaGS2-2Ab the source for
transport to different
allele have a higher grain nitrogen concentration. Intriguingly, transgenic expression of TaGS2–
sinks in plants
2Ab under the control of its own promoter in wheat resulted in higher grain yield. It should be
also noted that while GS genes certainly have an important role in nitrogen remobilization during CRISPR/Cas9:
senescence, other enzymes, such as proline dehydrogenase and glutamate dehydrogenases, could a simple yet powerful
tool for editing
also participate in nitrogen remobilization by providing an ammonium substrate.
genomes that easily
It is highly desirable to transport the most nutrient possible from senescing leaves to seeds. alters DNA sequences
Amino acid and peptide transporters that are up- or downregulated during leaf senescence could and modifies gene
be potential candidates. Arabidopsis AMINO ACID PERMEASE8 (AAP8) was shown to be ex- function
pressed in the phloem of source leaves, suggesting its function in phloem loading. In the aap8
mutant, decreased amino acid phloem loading and partitioning to sinks were observed, result-
ing in decreased silique and seed numbers (97). A potential role of AAPs in phloem loading and
nitrogen remobilization was also supported from studies in pea, where overexpression of the en-
dogenous AAP1 transporter in the leaf phloem led to increased source-to-sink allocation of amino
acids and improved seed yield (127).
Transcriptome analyses during leaf senescence have been performed with many crop plants.
However, most of the genes predicted to be involved in efficient nitrogen usage are functionally
uncharacterized. The CRISPR/Cas9-based genetic editing system and high-throughput screen-
ing with a noninvasive phenotyping system in the mutants, transgenic lines, or varieties are likely
to facilitate the identification of key molecules and the manipulation of those genes. As a comple-
mentary approach, analyzing the metabolic flux analysis during leaf senescence is likely to yield
further insights into the molecular mechanisms of nitrogen remobilization.
Manipulation of Key Regulatory Genes Associated with the External

and Internal Factors that Affect Leaf Senescence
Manipulation of leaf senescence exploiting key regulatory genes is primarily based on altering the
signaling or level of endogenous hormones and changing the expression of key TFs that control
SAGs.
Hormone-mediated strategy. Successful modulation of endogenous cytokinin levels was ob-

tained using an autoregulated senescence inhibitor systemin which the isopentenyl transferase
coding gene, IPT, is fused to the SAG12 promoter of Arabidopsis. This strategy was successfully
applied in many plant species including important agronomic crops, such as rice, wheat, and let-
tuce, and resulted in a higher seed set and in the accumulation of more biomass (34). It should be

noted that prolonged leaf longevity may prevent effective nutrient recycling. This may be in good
agreement with the fact that, in many crop plants, stay-green cultivars usually show higher grain
yield, but nitrogen remobilization in these cultivars is lower than expected. A balance between de-
layed leaf senescence and nutrient translocation to sink tissues is probably needed for increasing
yield in crops (34).
Ethylene is a representative hormone promoting leaf senescence. Research on the genetics of
ethylene biosynthesis has been successfully translated from Arabidopsis to crops and from the labo-
ratory to the field. For example, in maize, downregulation of ACS6 shows prolonged leaf longevity
when exposed to drought stress (123) and results in a significant increase in grain yield when ex-
posed to drought stress in the field (35, 99, 123). AUXIN-REGULATED GENE INVOLVED
IN ORGAN SIZE (ARGOS) proteins are negative regulators of ethylene response. Interestingly,
CRISPR/Cas9-engineered variants of maize with increased ZmARGOS8 expression levels show a
higher grain yield under drought stress conditions (99, 100).
Given the wide variety of ethylene-mediated developmental processes, disease and stress re-
sponses, and interaction with other hormone responses, genetic alteration of upstream members of
the ethylene-signaling pathway often results in pleiotropic phenotypes. Thus, downstream play-
ers, such as the ETHYLENE RESPONSE FACTORs, could be better candidates to target. The
overexpression of HIGHER YIELD RICE, a rice ERF, has increased shoot biomass and grain yield
under normal and drought conditions (3). Unraveling the highly complex regulatory networks
in which ERFs are involved will enable new advances in the targeted engineering of ethylene-
mediated responses.
Transcription factor–mediated strategy. Senescence-manipulating technology involving some

key senescence-specific TFs, such as AtNAP, appears very promising for crop improvement.
Attempts have been made to suppress the expression of the AtNAP homologous genes in a
variety of plant species (34). Analysis of the OsNAP-repressed transgenic lines shows a 24% in-
crease in rice grain yield. Similarly, suppression of the maize ortholog of AtNAP, ZmNAP, by RNA
interference (RNAi) silencing showed delayed leaf senescence and a 15–30% increase in thousand
grain weight (129). RNAi silencing of three NAP orthologs in wheat flag leaves caused a 24-day
delay in the senescence of flag leaves (112). The drastic delay of senescence in wheat, however, did
not lead to any increase in yield. Moreover, the RNAi plants showed a reduction in grain protein,
zinc, and iron content (112). Nutrient remobilization from senescing leaves critically contributes
to grain yield and quality, so an extended delay of leaf senescence may not be desirable in some
crops.
It is expected that different plant species will have different senescence physiology. Thus,
knowledge obtained from a model species may not be transferrable to other plant species. Compar-
ative study of the functions of the homologous regulatory genes of senescence among plant species
that have adapted in different environments may also reveal important aspects of the senescence
program.
SUMMARY POINTS
1. Complex transcriptional regulation involving diverse transcription factors (TFs), includ-
ing bZIP (ABI5 and EEL), NAC (ANAC019 and ORE1), and MYC (MYC2, 3, and 4)
family TFs, have uncovered several key regulatory mechanisms that control chlorophyll
breakdown during leaf senescence.
15.22 Woo et al.

2. Temporal dynamics and functions of gene regulatory networks involving the NAC and
WRKY TF families reveal important roles for these families in the regulation of leaf
senescence. Notable findings include the identification of key regulatory network mod-
ules involved in ethylene-mediated chlorophyll degradation and NAC TF hub molecules
responsible for time-dependent NAC network transitions.
3. A range of transcriptomic, epigenomic, proteomic, and metabolomic studies have im-
proved our current understanding of the functional and regulatory transitions in leaf
senescence.
4. Senescing leaves have tighter temporal coordination of transcriptomes than growing
leaves.
5. The regulatory programs of leaf senescence are finely regulated at multiple chromatin,
transcriptional, posttranscriptional, translational, and posttranslational levels by inte-

grating internal and external signals into the leaf senescence program.
6. The circadian clock is emerging as a key regulator of leaf senescence.

7. Autophagy is an important process for nitrogen recycling and remobilization during
senescence. Manipulation of the autophagy pathway is a promising strategy for the im-
provement of crop yields.
8. Senescence-manipulating technology utilizing the PSAG -IPT autoregulatory senescence
inhibition system, ethylene pathways, and AtNAP, one of key senescence regulatory TFs,
was successful at improving yields and the length of postharvest storage in many crop
plants, showing great potential for commercialization.
FUTURE ISSUES
1. Most of the previous studies on leaf senescence have been focused on the late stages of
aging. However, leaf senescence, as an integral part of plant development, is affected not
just by the current internal and external conditions, but by all of the earlier developmen-
tal stages. Thus, it may be better understood from a life history perspective.
2. Leaf senescence involves crucial time-dependent physiological changes and environ-
mental responses. Thus, the complex process of senescence might be better understood
in the context of molecular network dynamics. Multilayered interaction-based analyses
of senescence, including the dynamics of protein–protein, protein–DNA, protein–RNA,
and RNA–RNA complexes in a time-dependent manner or spatial networks that involve
mitochondria, chloroplasts, vacuoles, cytoplasm, and nuclei, are a crucial next step.
3. Integration of multi-omics data will decode spatiotemporal, age-associated networks.
Critically missing are proteomic data. In addition, the use of plant phenomics will be
required to investigate physiological changes along the entire lifespan of a leaf and to
elucidate age-associated changes in morphology, physiology, and molecular behaviors
in a comprehensive manner. Integrative analyses of multi-omics data sets will enable
the identification of key determinants in the developmental transition to the senescence
stage by mapping the modules whose temporal changes are mostly correlated with those
of physiological, molecular, and metabolic characteristics.

4. Senescence is regulated by age. How age information is perceived and how it is processed
are fundamental questions for enhancing our understanding of leaf senescence. Studies
on the interaction of age information with the circadian clock are a first step toward
answering this question.
5. Arabidopsis leaves show a vast variety of leaf senescence phenotypes. Evolutionary mech-
anisms associated with reproduction may be pursued utilizing these ecotypes. High-
throughput phenomic analysis of various physiological and developmental traits from
Arabidopsis accessions will facilitate the evaluation of each trait with respect to its impor-
tance for fitness.
6. The mechanisms observed in Arabidopsis need to be compared with those in crop plants
and further compared with those in animals.
7. Genetic resources and knowledge for manipulating leaf senescence have been rapidly ex-
panding. Because leaf senescence involves the spatial and temporal regulation of complex
networks, a finer approach that includes the spatial and temporal modification of gene
expression will be needed. Approaches such as the CRISPR/Cas9 system for genome
editing should help toward this goal.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank the many colleagues who have contributed to leaf senescence research and apolo-
gize to those whose work was not included owing to space limitations. This research was sup-
ported by the Institute for Basic Science (IBS-R013-D1) and the Mid-Career Researcher Program
(2017R1A2B4012937 and 2018R1A2A3075033) of the National Research Foundation of Korea
(NRF) through funding from the Korean Ministry of Science and ICT (MSIT).
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cls February 16, 2019 14:4

Annual Review of Plant Biology

and Hong Gil Nam1,2,†

Annu. Rev. Plant Biol. 2019. 70:15.1–15.30 Keywords

Copyright © 2019 by Annual Reviews. Abstract

The Dynamics and Functions of WRKY Gene Regulatory Networks Underlying

Omics Approaches for Temporal Profiling of Molecular Processest

15.2 Woo et al.

MULTIFACETED CHANGES IN CHLOROPLASTS AND

Regulation of Chlorophyll Breakdown and Chloroplast

www.annualreviews.org • Leaf Senescence 15.3

Cell wall biogenesis Cell wall disassembly

Chromatin assembly/DNA replication

b ANAC083 ANAC017 ANAC082 ANAC090 ANAC055 ANAC090 ANAC076 ANAC079

(Caption appears on following page)

15.4 Woo et al.

Figure 1 (Figure appears on preceding page)

breakdown is governed by the pheophorbide a oxygenase (PAO)/phyllobilin pathway, which

by NONYELLOW COLORING1 (NYC1) and NONYELLOW COLORING1–LIKE (NOL), and

www.annualreviews.org • Leaf Senescence 15.5

NYC1 NOL NYE1/SGR1 NYE2 PPH PAO RCCR

Chlorophyll b Chlorophyll a Pheophytin a Pheophorbide a RCC pFCC

15.6 Woo et al.

branched structures that are formed by interconnected mitochondria to enlarged, round-shaped

THE FUNCTIONAL AND REGULATORY TRANSITIONS

The Temporal Dynamics and Functions of Gene Regulatory Networks

www.annualreviews.org • Leaf Senescence 15.7

The Dynamics and Functions of WRKY Gene Regulatory Networks Underlying

15.8 Woo et al.

Chromatin-mediated regulation Ac H3Ac

ERF4/8 WRKY57 WRKY57

Activation Suppression Change or modification Transcription

15.10 Woo et al.

late leaf senescence.

MULTILAYERED REGULATION OF STRESS-INDUCED LEAF

The Chromatin-Mediated Regulation of Stress-Induced Leaf Senescence

www.annualreviews.org • Leaf Senescence 15.11

mutant of an Arabidopsis histone acetyltransferase gene, HOOKLESS1 (HLS1), leads to enhanced

The Transcriptional Regulation of Stress-Induced Leaf Senescence

15.12 Woo et al.

Posttranscriptional Regulation of Stress-Induced Leaf Senescence

supporting that differential expression of alternatively spliced transcripts might be an important

Posttranslational Regulation of Stress-Induced Leaf Senescence

www.annualreviews.org • Leaf Senescence 15.13

CROSSTALK BETWEEN HORMONE SIGNALING

15.14 Woo et al.

ICS1 CRF6 CRF6

ANT SA-response genes (PR1) Cytokinin-response genes

Dark COI1 AAO3

JAZ7 JAZ4/8 WRKY57

SAG29 CCGs (NYE1, NYC1, PAO)

WRKY45 WRKY75 WRKY46/51/63

SAG12/SAG113 SAG13/SGR CAT2 SID2

Activation Suppression Transcription

www.annualreviews.org • Leaf Senescence 15.15

Figure 4 (Figure appears on preceding page)

ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR; AtNAP, NAC-LIKE, ACTIVATED BY AP3/PI; BFN,

15.16 Woo et al.

www.annualreviews.org • Leaf Senescence 15.17

transcriptional regulation of GA-promoted leaf senescence in Arabidopsis.

OTHER EMERGING REGULATORY MECHANISMS OF LEAF

Light Signaling and Leaf Senescence

15.18 Woo et al.